The global number of patients with thromboses such as myocardial infarction, cerebral infarction and peripheral artery occlusive disease is very large, and these diseases are very significant diseases to be diagnosed and treated. Platelets play a fundamental role for the onset of these thromboses. In general, if vascular endothelial cells in blood vessel cavities are impaired by arteriosclerotic lesion or the like, platelets will adhere to the impaired region to cause activation, and thus there are formed thrombocytic thrombi, which eventually develop into occlusive lesions.
As one of the methods for detecting activation of platelets, there is a method of measuring glycocalicin concentration in plasma. Glycocalicin is a protein consisting of an enzymatically cleaved extracellular portion of a membrane glycoprotein present on surfaces of platelets, glycoprotein Ibα chain, and has a molecular weight of about 135 kDa. It is known that glycocalicin concentration in plasma is increased by impairment or activation of platelets, and it is currently used as a marker for detecting presence or absence of thrombotic diseases in clinical diagnosis. (J. H. Beer et al., Blood, 83, 691-702, 1994; S. Kunishima et al., Clin. Chem., 37, 169-172, 1991).
Many measurement methods of glycocalicin concentration have been reported, and any of these are based on ELISA (enzyme-linked immunosorbent assay) technique, wherein glycocalicin is detected by the sandwiching method utilizing two kinds of monoclonal antibodies directed to glycocalicin (J. H. Beer et al., supra; S. Kunishima et al., supra). Briefly, first monoclonal antibodies are immobilized on a 96-well plate or the like as a solid phase, blocked with a protein such as bovine serum albumin (BSA), and then added with patient's plasma (or serum) to be measured. Glycocalicin specifically binds to the monoclonal antibodies immobilized on the solid phase. The plate is washed, and added with second monoclonal antibodies labeled with an enzyme such as alkaline phosphatase and peroxidase or biotin so that the second antibodies should specifically bind to the glycocalicin bound to the first monoclonal antibodies. After washing, the plate is added with a substrate that can be converted into a substance exhibiting specific absorbance in a UV or visible region, fluorescence or luminescence with the enzyme used as the label of the second antibodies to perform an enzymatic reaction. Since the amount of glycocalicin in the patient's plasma and the binding amount of the second antibodies show positive correlation, the concentration of glycocalicin in the patient's plasma can be measured by quantifying the reaction product produced by the enzymatic reaction. A measurement method for glycocalicin by competitive ELISA utilizing one kind of anti-glycocalicin antibodies has also been reported (H. Bessos et al., Thromb. Res., 59, 497-507, 1990). However, the IC50 value of the glycocalicin concentration showing competitive inhibition is about 4 μg/ml, and this makes the above measurement unusable for the measurement of the glycocalicin concentration in plasma (it is about 2 μg/ml in a healthy subject, J. H. Beer et al., supra).
The aforementioned glycocalicin quantification methods based on the sandwich technique are widely used at present. However, when a similar measurement system is desired to be newly prepared, it is necessary to obtain two kinds of anti-glycocalicin monoclonal antibodies having different recognition sites. Commercially available monoclonal antibodies are generally very expensive, and the preparation of monoclonal antibodies requires much labor such as acquisition of glycocalicin for immunization, acquisition of hybridoma from a spleen of immunized mouse and screening of a monoclonal antibody-producing cell. Further, it is impossible to measure an absolute value of glycocalicin concentration from the amount of the enzymatic reaction in the aforementioned sandwich ELISA method, and in many cases, it is necessary to measure glycocalicin of several kinds of known concentrations to obtain a calibration curve, and then it is necessary to calculate a concentration in a test sample to be measured based on comparison with the calibration curve. Therefore, it is important to establish a method capable of measuring an absolute concentration of glycocalicin in a simple manner without the complicated preparation of monoclonal antibodies, from a viewpoint of wide use in clinical diagnosis.
Further, in an early stage of onset of thrombosis, von willebrand factor in blood binds to subendothelial tissues (collagen etc.) exposed due to impairment of vascular endothelial cells, and the membrane glycoprotein, glycoprotein Ib, on platelets binds to the von Willebrand factor. Thus, the platelets adhere to blood vessel walls, and they are activated (J. P. Cean et al., J. Lab. Clin. Med., 87, 586-596, 1976; K. J. Clemetson et al., Thromb. Haemost., 78, 266-270, 1997). Therefore, it is an important target of antithrombotic drugs for treating or preventing thromboses to inhibit the binding of von Willebrand factor and glycoprotein Ib. However, there are few substances that have been proven to exhibit antithrombotic property by inhibiting the binding of the both proteins.
It has been reported that a recombinant protein VCL that has a sequence of from the 504th to 728th amino acid residues of von Willebrand factor shows an antithrombotic action by inhibiting the binding of von Willebrand factor and glycoprotein Ib (K. Azzam et al., Thromb. Haemost., 73, 318-323, 1995). Further, it has also been reported that a monoclonal antibody AJvW-2 directed to human von Willebrand factor exhibits an antithrombotic activity by specifically binding to von Willebrand factor without showing hemorrhagic tendency (S. Kageyama et al., Br. J. Pharmacol., 122, 165-171, 1997; WO 96/17078). Furthermore, the protein AS1051 derived from snake venom specifically binds to the platelet glycoprotein Ib to similarly exhibit an antithrombotic property without showing hemorrhagic tendency (N. Fukuchi et al., WO 95/08573).
Further, aurintricarboxylic acid (ATA), which is a pigmental compound, has been reported to show an activity for inhibiting the binding of von Willebrand factor and glycoprotein Ib (M. D. Phiillips et al., Blood, 72, 1989-1903, 1988). However, it is known that its binding specificity is not high (K. Azzam et al., Thromb. Haemost., 75, 203-210, 1996; D. Mitra et al., Immunology, 87, 581-585, 1996; R. M. Lozano et al., Eur. J. Biochem., 248, 30-36, 1997), and that the inhibition activity is exhibited by a polymerized macromolecule fraction (M. Weinstein et al., Blood, 78, 2291-2298, 1991; Z. Gua et al., Thromb. Res., 71, 77-88, 1993; H. Matsuno et al., Circulation, 96, 1299-1304, 1997) etc.
As described above, although it is an important target of antithrombotic drugs to inhibit the binding of von Willebrand factor and glycoprotein Ib, there is no low molecular weight compound that has reported to inhibit the binding of the both and have an antithrombotic activity, and therefore it is important to find out such a substance for attempting treatment and prevention of thromboses.
As a non-proteinaceous substance that inhibits the binding of von Willebrand factor and glycoprotein Ib, aurintricarboxylic acid (ATA) can be mentioned. However, it is known that it exhibits the inhibitory activity as a polymerized macromolecular substance as already described above. M. Weinstein et al. (Blood, 78, 2291-2298, 1991) investigated an activity of ATA fractionated by gel filtration for inhibiting the ristocetin-induced aggregation, which is von Willebrand factor and glycoprotein Ib dependent platelet aggregation, and concluded that a polymer having a molecular weight of 2500 had the strongest activity. They also showed that fractions eluted as low molecular weight fractions in the gel filtration scarcely have the activity (FIGS. 1 and 3 in the aforementioned reference). Moreover, in this report, neither a specific structure nor molecular weight of the ATA polymer showing the activity was specified. It is considered that there are no ATA derivatives exhibiting the inhibitory activity for the binding of von Willebrand factor and glycoprotein Ib among those of which structure can be specified, in view of the facts that, although the synthesis of the ATA monomer has already been reported by R. D. Haugwitz (WO 91/06589), no data were reported so far for demonstrating the inhibition of the binding of von Willebrand factor and glycoprotein Ib as for the monomer or a polymer of which structure can be specified, and evaluation of the activity has been reported by using a gel filtration fraction of ATA polymer even in a recent study (T. Kawasaki et al., Amer. J. Hematol., 47, 6-15, 1994).
In the aforementioned report by M. Weinstein et al. (Blood, 78, 2291-2298, 1991), it is described that presence of many negative electric charges (polyanion) and presence of many aromatic rings (polyaromatic) are necessary for the inhibition of the binding of von Willebrand factor and glycoprotein Ib. The fact that the abundance of negative electric charges is likely to inhibit the binding of von Willebrand factor and glycoprotein Ib is also consistent with the fact that heparin, which is a polysaccharide having negative electric charges, inhibits the binding of von Willebrand factor and glycoprotein Ib (M. Solbel et al., J. Clin. Invest., 87, 1787-1793, 1991). In this report, it is also reported that the activity for inhibiting the binding of von Willebrand factor and glycoprotein Ib is reduced, as the molecular weight of heparin becomes smaller.
Heparin is originally a substance inhibiting thrombin, which is a blood aggregation factor, and the blood aggregation factor X (factor Xa). Although a heparin derivative that was imparted with higher selectivity for the binding of von Willebrand factor and glycoprotein Ib has also been reported (M. Sobel et al., Circulation, 93, 992-999, 1996), the average molecular weight of that substance is 10,000 or more.
Among substances that are likely to bind to proteins, there is also reported a substance that shows the selective inhibitory activity to some extent for the binding of von Willebrand factor and glycoprotein Ib. It was demonstrated that Evans Blue, which is a pigmental compound, selectively inhibited the platelet aggregation in which von Willebrand factor (factor VIII in this reference) was involved (E. P. Kirby et al., Thrombos. Diathes. Haemorrah., 34,770-779, 1975). However, the experimental results contained in this reference all concerned platelet aggregation under a condition not containing blood plasma, and no reference was made for the activity under a condition where plasma proteins are present. Evans Blue is originally a substance that very firmly binds to serum albumin, and such a property provides its use as means for measurement of blood volume, blood leak from blood vessels in living bodies and so forth (M. Gregersen & R. A. Rawson, Physiol. Reviews, 39, 307, 1959). That is, when treatment of living bodies, for example, humans, is intended, such substances that strongly bind to proteins in plasma as mentioned above would not show the effect at all. As such substances, there are sulfobacin (T. Kamiyama et al., J. Antibiot., 48, 924-928, 1995) and so forth. Although sulfobacin showed the specificity for the binding of von Willebrand factor and glycoprotein Ib to some extent according to the above reference, it must not show the activity due to the binding to plasma proteins in blood or blood plasma in view of its detergent-like structure. In fact, its inhibitory activity for the platelet aggregation in plasma was not shown in the aforementioned reference.
As described above, any low molecular compounds have not been known so far, which can inhibit the binding of von Willebrand factor and glycoprotein Ib in living bodies. Assuming drugs against thrombotic diseases for inhibiting the binding of von Willebrand factor and glycoprotein Ib, if they are used as an injection, they may be a macromolecular compound such as proteins or polymers. However, in order to create a drug of the same mechanism of action that can be orally administered, it is important to find a low molecular weight substance that is not a polymer and completely and selectively inhibits von Willebrand factor and glycoprotein Ib dependent platelet aggregation in blood (in plasma).
However, such compounds have not been found so far. As a reason for this, there can be mentioned the fact that any assay system enabling screening of such a substance in a simple manner has not existed.
As will be described later, in conventionally used assay methods for detecting the binding of von Willebrand factor and glycoprotein Ib, 125I-labeled von Willebrand factor are bound to platelets or formalin-fixed platelets. However, such methods suffer from complexity of using the radioactive isotope, and difficulty of obtaining a large amount of sample, i.e., difficulty that blood must be collected from an animal, and platelets must be obtained from it. The methods generally used so far and means for solving the problems thereof will be specifically described below.
The binding of von Willebrand factor and glycoprotein Ib is not observed under a usual condition, and it is considered that it occurs only under a condition where shear stress is induced in a blood flow (T. T. Vincent et al., Blood, 65, 823-831, 1985). However, as a method for artificially making it possible to observe the binding of the both proteins, there are known addition of an antibiotic, ristocetin (M. A. Howard and B. G. Firkin, Thromb. Haemost., 26, 362-369, 1971), and addition of a protein derived from snake venom, botrocetin (M. S. Read et al., Proc. Natl. Acad. Sci. USA., 75, 4514-4518, 1978). That is, the both substances are known as a substance that binds to a specific site of von Willebrand factor to cause a structural change of the von Willebrand factor, thereby causing the binding of the von Willebrand factor and glycoprotein Ib, which does not occur under a usual condition. As a method for observing the binding of the both proteins, there is the following method reported by Fujimura et al. (Y. Fujimura et al., Blood, 77, 113-120, 1991).
That is, human von Willebrand factor is labeled with 125I in a conventional manner, and allowed to bind to formalin-fixed platelets in the presence of a certain amount of ristocetin or botrocetin. This binding occurs due to the specific binding of the von Willebrand factor to glycoprotein Ib on the surfaces of the immobilized platelets, and after unbound von Willebrand factor are removed by washing, the amount of the both proteins bound to each other can be measured by measuring the amount of 125I. Miura et al. detected the binding of the both proteins by a similar method, wherein platelets were immobilized on a 96-well plate via immobilized anti-platelet membrane protein antibodies instead of the use of formalin-fixed platelets (S. Miura et al., Anal. Biochem., 236, 215-220, 1996). Further, Matsui et al. reported a method of binding glycocalicin, which is a partial protein of the extracellular portion of glycoprotein Ibα chain in the presence of botrocetin, to von Willebrand factor bound to collagen immobilized as a solid phase (T. Matsui et al., J. Biochem., 121, 376-381, 1997). Furthermore, Moriki et al. produced a recombinant protein expressing cell that expressed glycoprotein Ib on the membrane, and reported that 125I-labeled von Willebrand factor bound to the glycoprotein Ib on the membrane in the presence of botrocetin. Moriki et al. further produced a cell expressing glycoprotein Ib having a mutation in the amino acid sequence, which bound to von Willebrand factor without any inducing agent, and performed a binding experiment. However, the binding amount was very small compared with the binding amount in the presence of botrocetin or ristocetin (T. Moriki et al., Blood, 90, 698-705, 1997).
As described above, all of the methods reported so far for detecting the binding of von Willebrand factor and glycoprotein Ib with high sensitivity are methods by obtaining a large amount of platelets or glycoprotein Ib expressing cells and detecting the binding of von Willebrand factor to them. However, it is extremely laborious to routinely prepare a large amount of platelets or such cells, and therefore it is necessary to develop a method for detecting the binding of von Willebrand factor and glycoprotein Ib in a simpler manner.
Further, all of the conventionally used methods are exclusively methods utilizing addition of a binding inducing substance such as botrocetin or ristocetin to a liquid phase. However, the amount of botrocetin or ristocetin changes the amount of the binding of von Willebrand factor and glycoprotein Ib. Moreover, if a large number of binding experiments are performed by using a 96-well plate, for example, these methods utilizing addition of the inducing substance to the liquid phase are laborious. Furthermore, when the aforementioned low molecular weight substance inhibiting the binding of von Willebrand factor and glycoprotein Ib is searched, an extremely large number of binding experiments must be performed, and therefore it is also important from this viewpoint to solve the aforementioned problem.
As already stated, a true inhibition substance with low molecular weight for the binding of von Willebrand factor and glycoprotein Ib has not been discovered yet. The term “true inhibition substance” used herein means a substance specifically inhibiting the binding of von Willebrand factor and glycoprotein Ib, but does not mean a substance that inhibits the binding of von Willebrand factor and glycoprotein Ib in a non-specific manner, even though it may inhibit the binding of von Willebrand factor and glycoprotein Ib, like substances that generally change structures of proteins such as protein denaturing substances and detergents or substances that non-specifically bind to proteins.
As described above, such true inhibition substances for the binding of von Willebrand factor and glycoprotein Ib have been found among antibodies, proteins derived from snake venom, pigmental substances such as aurintricarboxylic acid (ATA), of which active body is a substance having a high molecular weight. However, no such substance has been known among low molecular weight substances, for example, those having a molecular weight of 2000 or less, in particular, those having a molecular weight of 1000 or less, which are useful for oral administration. Therefore, it has been desired to develop an evaluation system capable of quickly screening such substances.